Phosphorolytic activity of Escherichia coli glycyl‐tRNA synthetase towards its cognate aminoacyl adenylate detected by 31P‐NMR spectroscopy and thin‐layer chromatography

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Abstract
The catalytic activity of highly purified E. coli glycyl-tRNA synthetase was studied by 31P-NMR spectroscopy and TLC on poly(ethyleneimine)-cellulose. Besides the activation of its cognate amino acid and the syntheses of adenosine(5'')tetraphospho(5'')adenosine (Ap4A) and adenosine(5'')triphospho(5'')adenosine (Ap3A), this synthetase also catalyzes the formation of ADP from Pi and the enzyme-bound glycyl adenylate. In the presence of Pi, glycine and Mg2+, the enzyme catalyzes the synthesis of ADP from 3 different substrates which all lead to enzyme-bound glycyl adenylate, i.e., ATP, adenosine 5''-[.beta.,.gamma.-methylene]triphosphate and Ap4A. The only pathway by which a synthetase-actalyzed degradation of Ap4A can occur is through the reaction between Pi and the enzyme-bound glycyl adenylate, synthesized from Ap4A. A 20-fold increase of the phosphorolytic activity of the synthetase is observed when Mg2+ is replaced by Mn2. Besides establishing the phosphoryltic activity of the enzyme, the study also showed that the preparation catalyzes a glycine-independent transfer of the .gamma.-phosphate group from ATP nucleoside 5''-diphosphates. The importance of the reaction between Pi and enzyme-bound aminoacyl adenylate in relation to the remaining catalytic activities of aminoacyl-tRNA synthetases is discussed, as is the biological significance of the reaction.

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